This project started over 35 years ago to isolate, characterize and define cell functions for molecules controlling actin filament (F-actin) remodeling responsible for mammalian cell motility and phagocytosis. In the 1970s, we discovered two proteins contributing to cytoplasmic "gel-sol transformations," filamin A (FLNa), which gels F-actin and gelsolin, which regulates this gelation by reversible F-actin severing. Although filamin A and gelsolin are clearly important for cell motility, and their deficiency or mutation lead to human diseases, with time these proteins have become imbedded within hundreds of others contributing to actin remodeling, including filamin and gelsolin isoforms. We have therefore increasingly focused on the structure and function of filamin A. During the current grant cycle, our ability to generate full-length, truncated and domain-swapped FLNa proteins helped clarify how FLNa orients to specifically and potently cross-link F-actin. It also facilitated the discovery of calcium regulation of F-actin binding by FLNa through calmodulin and of unsuspected F-actin binding regions in FLNa subunits. We demonstrated that F-actin binding by FLNa slows F-actin turnover in vitro and in vivo. We also identified that FLNa subunit flexibility, conferred in part by a surprisingly small hinge sequence, enables pre-stress to cause FLNa-F-actin gels to achieve high stiffness values associated with living substrate-attached cells. We also showed that myosin II filaments within FLNa-F-actin gels impose pre- stress, introducing a unique biomimetic machine. The multi-domain contributions of FLNa to F-actin mechanics and dynamics pale in complexity before the scores and probably hundreds of binding partners interacting with and functionally influenced by FLNa, including receptors, signaling intermediates, enzymes and transcription factors. We believe that only atomic structural information on FLNa and its interfaces with binding partners to inform point mutant reagents will enable us to sort out its cellular functions with confidence and think about designing inhibitors to treat diseases. We therefore now collaborate with structural biologists to solve FLNa atomic structures and prove concept that point mutations selectively impair specific FLNa functions, leaving most others intact. We also showed how FLNa's substructure accommodates both its F-actin cross-linking and partner binding and suggested how domain unfolding might regulate partner binding. We propose to build on this strategy, solving additional FLNa substructures as well as complexes with selected representative partners: CXCR4, CFTR and FilGAP, a Rac GAP we discovered that determines cell polarity. We will use point mutants to study the regulation of FilGAP in vivo and also investigate its role in neutrophil oxidase activity as well as obtain additional evidence for FLNa domain unfolding in partner binding regulation. PUBLIC RELEVANCE: This research concerns certain machinery parts in living cells that control how cells move, change shape, multiply and undertake many other functions. When these parts are missing or altered by genetic mutations, depending on the abnormality, mild or severe diseases occur. The research plan is to define the chemistry of one of these parts in sufficient detail to enable future development of drugs to ameliorate such diseases.